Leishmania organisms are intracellular protozoan parasites of macrophages that cause a wide range of clinical diseases in humans and domestic animals, primarily dogs. The life cycles of Leishmania involves a vertebrate host (e.g., a human) and a vector (a sand fly) that transmits the parasite between vertebrate hosts. In the vector the parasite takes on a characteristic morphological form known as the promastigote, and reproduces asexually in the vector's gut. When the vector bites a vertebrate host, promastigotes are injected into the host. The promastigotes then enter cells of the vertebrate host and change into a form known as the amastigote. The amastigote reproduces in the host's cells and, when the cells eventually die, the amastigotes are released and infect other cells. The symptoms and pathology associated with leishmaniasis result from the amastigotes killing the host's cells.
In some infections, the parasite may lie dormant for many years. In other cases, the host may develop one of a variety of forms of leishmaniasis. For example, leishmaniasis may be manifested as a cutaneous disease, which is a severe medical problem. Several Leishmania species are responsible for cutaneous leishmaniasis. In the Middle East and Central Asia the predominant species responsible for cutaneous forms of leishmaniasis are L. major and L. tropica, with L. donovani and L. infantum predominantly leading to visceral forms of leishmaniasis. In Iraq L. major is the major cause of cutaneous leishmaniasis with most of the reported cases in the army being due to this agent. L. major is the primary agent for zoonotic cutaneous leishmaniasis (ZCL). In Afghanistan the primary agent for cutaneous leishmaniasis is L. tropica with an active infection rate in Kabul of 2.7%. However areas of Northern Afghanistan are also endemic for L. major. L. tropica is more frequently associated with anthroponotic cutaneous leishmaniasis (AZL). With the deployment of U.S. troops to the Middle East and Central Asia there has been a significant increase in the number of personnel developing cutaneous leishmaniasis, particularly in Iraq. This has raised the need for a field test to identify the presence of Leishmania parasites directly in skin lesions.
Current diagnostic procedures are not readily applicable to field situations and typically require centralized laboratory testing. In particular PCR has gained a forefront in testing for Leishmania species involved in cutaneous leishmaniasis. Material from skin scrapings or biopsy is obtained, and DNA extracted and subjected to PCR analysis. New PCR methods are being developed to enable multiple Leishmania species to be detected and differentiated in a single assay. Other methods used for identifying parasites include culture from skin biopsy samples, which is time consuming, or light microscopic analysis or histology of thin tissue sections from lesions or biopsies to analyze for the presence of parasites. In the case of visceral leishmaniasis, a rapid diagnostic test is available based on use of the repeat antigen K39 to detect the presence of antibodies to L. donovani, L. chagasi and L. infantum in serum (Scott et al., Am. J. Trop. Med. Hyg., 44:272-277, 1991). A similar assay does not currently exist for Leishmania species involved in cutaneous leishmaniasis.
Serodiagnosis looking for specific antibodies in cutaneous leishmaniasis has been attempted with some success using secreted antigens but this test lacks sufficient sensitivity and specificity. More recently, less invasive procedures have been used involving aspirates or scrapings from skin lesions or by using swabs (see, for example, Matsumoto et al., Trans. R. Soc. Trop. Med. Hyg. 93:606-7, 1999). These procedures lend themselves for use as collection devices for rapid field tests. Rabbit anti gp63 has been used in an indirect immunofluorescence assay for detection of Leishmania promastigotes and amastigotes in lesion aspirates (Mohareb et al., J. Egypt Soc. Parasitol. 28:313-321, 1998). Analysis of Western blot banding patterns has been used in the differential diagnosis of American cutaneous leishmaniasis (Goncalves et al., Am. J. Trop. Med. Hyg. 66:91-102, 2002). Skin tests using Leishmanin have also been used to evaluate latent infection but have generally been based on crude lysate antigen preparations and lack specificity (Arana et al., Trans. R. Soc. Trop. Med. Hyg. 93:394-6, 1999).
There thus remains a need in the art for a rapid and effective diagnostic test for cutaneous leishmaniasis that may be readily employed in a field situation.